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Journal of Chromatography B, 846 (2007) 105–111

Development and evaluation of an efficient HPLC/MS/MS method for thesimultaneous determination of pseudoephedrine and cetirizine in human

plasma: Application to Phase-I pharmacokinetic study

Ma Ming, Feng Fang ∗, Sheng Yulan, Cui Shuangjin, Liu HanDepartment of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China

Received 1 April 2006; accepted 18 August 2006Available online 12 September 2006

bstract

A sensitive, simple and highly selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and evaluatedo determine simultaneously the concentrations of pseudoephedrine and cetirizine in human plasma. The chief benefit of the present method is the

inimal sample preparation, as the procedure is only one-step protein precipitation. Two drugs were separated on a C8 column and analyzed byC/MS/MS using positive electrospray ionisation (ESI). The method had a chromatographic run time of 12.0 min and a linear calibration curvever the concentration range of 1.0–800 ng/ml for pseudoephedrine and 1.0–400 ng/ml for cetirizine, respectively. The lower limit of quantification

f the two drugs was 1.0 ng/ml, respectively. The intra- and inter-batch precisions were less than 9.7%. The method described herein has beenrst used to reveal the pharmacokinetic characters in healthy Chinese volunteers treated with oral administration of different dosages of cetirizineihydrochloride and controlled-released pseudoephedrine hydrochloride compound tablet, and approached the influence of a standard meal on thextent and rate of absorption of the combination tablet.

2006 Elsevier B.V. All rights reserved.

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eywords: Pseudoephedrine; Cetirizine; HPLC–MS/MS; Human plasma; Phar

. Introduction

Cetirizine dihydrochloride, is the carboxylated metabolitef hydroxyzine, and has high specific affinity for histamine1-receptors. Pseudoephedrine hydrochloride, which is a sym-athomimetic drug acts directly on alpha-adrenergic receptors.

Extended-release of pseudoephedrine/cetirizine tablet for-ulations twice- or once-daily have been marketed in the Bel-

ium since 1997. The twice-daily tablet consists of cetirizineihydrochloride 5 mg in an immediate-release coating and pseu-oephedrine hydrochloride 120 mg, of which 60 mg is in anmmediate-release coating and 60 mg is in a barrier-protectedore. To reveal the pharmacokinetic characters of two compo-ents at the combinatorial condition and direct the reasonable

sage of the drug, an effective, accurate and rapid method toetermine both cetirizine and pseudoephedrine in biologic flu-ds should be developed.

∗ Corresponding author. Tel.: +86 25 83271301.E-mail address: fengfang1@hotmail.com (F. Feng).

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570-0232/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.jchromb.2006.08.026

kinetics

To date, no analytical methods have been reported for simul-aneous determination of the two drugs in pharmaceutical prepa-ations and in biological samples. Various analytical methodsave been reported to determine them, respectively.

Many methods exit for pseudoephedrine quantification inuman plasma including HPLC with ultraviolet (HPLC-UV)1,2] and GC [3]. A liquid chromatography–tandem mass spec-rometry [4,5] was reported to determine the plasma levelf pseudoephedrine, but the extraction recovery was all lesshan 80%. For cetirizine, HPLC-UV [6] and GC [7], TLC8] were reported. Recently, a hydrophilic interaction liquidhromatographic–tandem mass spectrometric method (HILIC-S/MS) [9,10] and a column switching HPLC method [11] were

eported to determine cetirizine in human plasma. The formerchieved a lower limit of quantization (LLOQ) of 1 ng/ml, buthe sample preparation was tedious, as solid phase extractionas used. The latter provided a LLOQ higher than 10 ng/ml,

nd the chromatographic run time for one sample was a littleonger (about 23 min).

Considering the huge numbers of the sample, building aimple, sensitive, rapid and accurate method to be convenient

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06 M. Ma et al. / J. Chroma

o pharmacokinetic research was necessary. In this study, aC–MS/MS method with selected-reaction monitoring (SRM)ode was first developed for simultaneous assay of the two drugs

n human plasma samples. This assay method was simple, sen-itive, stable and relatively rapid, without any tedious procedurepproached the LLOQ as the literature in the following section.

. Experimental

.1. Reagents and materials

Pseudoephedrine hydrochloride (98% purity), cetrizine dihy-rochloride (99% purity) and phenylalanine hydrochlorideinternal standard (I.S.)](>98% purity) were supplied by Jiangsunstitute for Drug Control (Nanjing, China). Methanol wasPLC/Spectro grade and purchased from Merck Company

Darmstadt, Germany). Other chemicals were all of analyticalrade and were used as received. Water was purified by redistil-ation before use.

.2. Instrumentation

A Thermo Finnigan TSQ Quantum Ultra tandem mass spec-rometer equipped with electrospray ionization (ESI) sourceSan Jose, CA, USA), a Finnigan surveyor LC pump and anutosampler was used for LC–MS/MS analysis. Data acquisitionas performed with Xcalibur 1.1 software (Thermo-Finnigan,an Jose, CA, USA). Peak integration and calibration were car-ied out using LC Quan software (Thermo-Finnigan).

.3. Chromatographic condition

A Lichrospher C8 column (5 �m, 250 mm × 4.6 mm i.d.,anbang Co., Huaian, China) was used for all of the chro-atographic separations. A two-solvent gradient elution was

erformed, with a flow rate of 1.0 ml/min. The solvent A wasater with 0.13% formic acid, and solvent B was methanol. Theobile phase composition started at 45% solvent A and 55%

olvent B, then was being increased linearly to 70% solvent Bn 2 min, and then being held for 8 min. The column was equili-rated at 45% solvent A and 55% solvent B for 2 min before theecond injection was initiated. The total period for one sampleas about 12 min. The column temperature was maintained at5 ◦C.

Mass spectrometric analysis was performed in the positiveon mode (ESI+) and set up in the selected reaction monitoringSRM) mode. Nitrogen was used as the sheath gas (35 psi) andhe auxiliary gas (5 psi). The capillary temperature was 350 ◦C,nd the spray voltage was 4000 V. Collision induced dissocia-ion (CID) studies were performed and argon was used as theollision gas with a collision cell gas pressure of 1.5 mTorr. Theollision energy was 18 eV. On the basis of the full-scan MSnd MS/MS spectra of the drug, the most abundant fragment

on was selected and the mass spectrometer was set to monitorhe transition m/z 166.04 → 147.99 for pseudoephedrine, m/z89.07 → 200.91 for cetirizine and 152.02 → 134.06 for inter-al standard. The scan time for each analyte was set to 0.5 s.

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B 846 (2007) 105–111

.4. Preparation of stock and sample solution

The stock solutions of pseudoephedrine and cetirizine wererepared by dissolving the accurately weighed reference com-ound in water to give a final concentration of 50 �g/ml sep-rately. The solutions were then serially diluted with water tobtain standard working solutions. Stock solutions of I.S. wasrepared in methanol at the concentration of 100 �g/ml andiluted to 80 ng/ml with methanol. All the solutions were thentored at 4 ◦C and were brought to room temperature before use.

.5. Sample preparation

0.8 ml I.S. solution (80 ng/ml) was added to 200 �l plasma in1.5 ml test tube. The samples were immediately vortexed formin and centrifuged at 3000 rpm (revolutions per minute) for0 min. 0.8 ml of the supernatant layer was transferred to anotherlean test tube, and centrifuged at 1,6000 rpm for another 10 min.wenty microlitres of the clean supernatant was directly injectednto the LC/MS/MS for analysis. The chromatographic eluentas diverted to waste for 1.0 min after each sample injection inrder to keep the ion source as clean as possible.

.6. Calibration and quality control samples

Calibration standard solutions were prepared by spikinglank human plasma with standard solutions to give concen-rations of 1.024/1.032, 2.048/2.046, 5.120/5.160, 10.24/10.32,1.20/25.80, 102.4/51.60, 204.8/103.2, 409.6/206.4, and19.2/412.8 ng/ml for pseudoephedrine and cetirizine. Qualityontrol (QC) samples, which were used both in pre-study val-dation and during the pharmacokinetics study, were preparedeparately to give concentrations of 2.048/2.048, 102.4/51.60,nd 819.2/412.8 ng/mL of pseudoephedrine and cetirizine.

.7. Method validation

The method was validated for selectivity, accuracy, precision,ecovery, calibration curve range, and stability according to theS FDA [12]. The selectivity was investigated by preparing and

nalyzing six individual human blank plasma samples at theLOQ.

Linearity was assessed by analyzing pseudoephedrine1.024–819.2 ng/ml) and cetirizine (1.032–412.8 ng/ml) inuman plasma. Calibration curves were analyzed by weightedinear regression (1/y, y: concentration of the analytes) ofssayed peak areas ratios versus nominal drug concentrations.

Accuracy and precision were assessed by determining QCamples at three concentration levels (five samples each) on threeifferent validation days. The precisions were determined as theSD (%), and the accuracies were expressed as percentages of

he nominal concentrations. The criteria used to assess the suit-bility of precision and accuracy was as follows: the RSD should

ot exceed 15% and the accuracy should be within 85–115%.

The absolute recoveries were evaluated for both pseu-oephedrine and cetirizine by comparing peak areas of therecipitated samples with peak areas of the direct injection of

togr. B 846 (2007) 105–111 107

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M. Ma et al. / J. Chroma

he pure authentic standard solutions dissolved in the methanolt three QC levels.

The stability of the analytes in stock solution was determinedy placing the stock solutions in the refrigerator (4 ◦C) for aeek. And the stability of the analytes in human plasma underifferent temperature and timing conditions was evaluated athree QC levels. The long-term stability was assessed after stor-ge of the test samples at −20 ◦C for five days. The thaw-freezetability was determined after five thaw-freeze cycles (−20 to0 ◦C) on consecutive days. The extraction storage stability wasssessed by placing QC samples being precipitated at −20 ◦C forve days and analyzed. The results were compared with thoseC samples freshly prepared, and the percentage concentrationerivation was calculated.

.8. Pharmacokinetic study

The validated LC–MS/MS assay was applied to Phase-I phar-aceutics study in five female and five male healthy adult

olunteers received oral administration. The study was approvedy the Ethics Committee. All the volunteers who were selectedfter completing a thorough medical, biochemical and physicalxamination gave informed consent after they were explainedhe aims and risks of the study.

The pharmacokinetic experiment was containing two partswo-way crossover designs containing oral administration ofingle-dosage and different fasted and fed states.

Crossover 1, after an overnight fast (10 h), the volunteers tookhe assigned tablet orally with 200 ml of water. The volunteersere treated with an oral dose of one tablet, and with an oralose of two tablets after 1 week washout period. Regular stan-ardized low-fat meals were not provided until 4 h after dosedministration; water intake was allowed after 2 h.

Crossover 2, the effect of a standard meal on the pharma-okinetics of one tablet was evaluated. After an overnight fast10 h), for fed treatment, volunteers took one tablet after a stan-ard breakfast whereas for the fasting treatment, volunteers tookne tablet without breakfast and continued to fast for 4 h more.here was a 7-day drug-free period between the two treatments.

Following the drug administration, venous blood samples5 ml) were collected into heparinized tubes according to the fol-owing schedule: immediately before administration and 0.17,.25, 0.33, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0, 13.0,4.0, and 34.0 h after dosing. Blood samples were centrifugedt 1500 g for 10 min to obtain the plasma. The plasma samplesere labeled and kept frozen at −30 ◦ until analysis.Pharmacokinetic parameters were determined from the

lasma concentration-time data. An analysis of log-transformednd non-transformed PK parameters was done using BAPP2rocedures.

. Results and discussion

.1. MS conditions selection

For the optimization of MS conditions, each of the ana-ytes and I.S. was directly introduced into MS detector using

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oephedrine mother ion: m/z 166.04; daughter ion: m/z 134.06. (B) Cetirizineother ion: m/z 389.07; daughter ion: m/z 200.91. (C) I.S. mother ion: m/z

52.02; daughter ion: m/z 134.06.

SI ionization and parameters such as sheath gas, the auxil-ary gas, collision induced dissociation and the collision energyere investigated to increase the detection sensitivity of pseu-oephedrine, cetirizine and I.S.

Because three of the compounds were of weak base, domi-antly protonated molecules [M + H]+ in full scan spectra at m/z66.04, m/z 389.07 and m/z 152.02 were observed. Fig. 1 dis-lays the structures and the mass spectrum of [M + H]+ ions from

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3.4.5. Recovery and stabilityTable 2 shows the recovery (extraction efficiency) of pseu-

doephedrine and cetirizine from human plasma following

Table 1Intra-, inter-batch precision and accuracy for analytes the in human plasma (inpre-study validation, n = 3 days, five replicates per day)

Concentration (ng/ml) RSD (%) Relative error (%)

Added Found Intra-batch Inter-batch

Pseudoephedrine2.048 2.000 6.65 9.70 2.34

102.4 107.4 4.67 5.39 4.88819.2 811.8 2.46 3.78 0.90

08 M. Ma et al. / J. Chroma

hree compounds. Pseudoephedrine gave an intense product iont m/z 147.99, formed by losing [−OH]. Cetirizine showed anntense ion at m/z 200.91 corresponding to the loss of [CHPh)Cl] group from [M + H]+ ion. Phenylalanine (I.S.) showedmajor fragment ion at m/z 134.06 corresponding to a neutral

oss of water. These major fragment ions at m/z 147.99, m/z00.91 and m/z 134.06 were chosen in the SRM acquisition forseudoephedrine, cetirizine and I.S., respectively.

.2. Chromatographic conditions optimization

It is critical to optimize chromatographic conditions to obtainood selectivity, high sensitivity, quick speed and symmetricaleak shape. Various compositions of mobile phase were there-ore, tried ae first by varying the percentages of organic solvent.t was found that under isocratic elution mode, an inverted peaklways existed just before that of pseudoephedrine and that theeak made the baseline separation difficult, therefore, accurateeasurement of pseudoephedrine area is impossible. To find the

ause and avoid the overlaying of the two peaks, different vol-mes of methanol (as protein precipitant) or proper amounts oformic acid were added to black plasma to match its compositiono the mobile phase,in a better manner but little improvement wasbserved. The disappearance of the inverted peak when watertead of blank plasma was used as analyte suggests that ana-ytes coming from plasma contain certain kinds of compoundshat reduce MS response. Various gradient conditions were thenxperimented and the best combination was selected to get base-ine separation and symmetry peak shape.

.3. Preparation of plasma samples

de Jager et al. [13] reported that protein precipitation for ceti-izine can get a more satisified recovery than that of liquid–liquidxtraction and that the latter was complex and time-consuming1,14]. In this study, a one-step protein precipitation procedureas adopted to simplify the sample preparation and the treat-ent could provide LLOQ lower than 1.0 ng/ml for both of the

nalytes. The selected protein precipitant was methanol becausef satisfactory efficiency in precipitating and less ion suppres-ion compared with those observed with acetonitrile, ethanolnd acetone.

.4. Method validation

.4.1. SelectivityThe LC/MS/MS method has high specificity because only

fragment ion derived from the [M + H]+ ion of the analytesf interest was monitored. The selectivity toward endogenouslasma matrix components was assessed in six different batchesf human plasma samples by analyzing blanks and spiked sam-les at LLOQ levels. Endogenous peaks at the retention time ofhe analytes were not observed for any of the plasma batches

valuated. This indicated no significant direct interference inhe SRM channel for the analytes at the expected retentionime. Fig. 2 shows the typical chromatograms of a blank plasmaample, a blank plasma sample spiked with pseudoephedrine,

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B 846 (2007) 105–111

etirizine at the LLOQ and I.S., and a plasma sample from aealthy volunteer 2 h after an oral administration. Typical reten-ion times for pseudoephedrine, cetirizine and I.S. were 3.2, 7.7,nd 3.2 min, respectively. The total run time was about 12 min.

.4.2. Matrix effectsMatuszewski et al. [15] reported that matrix components,

hich co-elute with analytes, may adversely affect the repro-ucibility of analyte ionization in a mass spectrometer’s elec-rospray source. For pseudoephedrine, cetirizine and I.S., the

ean peak area from the six different samples sources had rel-tive error of 2.3–5.6%, 3.6–4.8% and 3.2–5.1%, respectivelyompared with those from standard solutions. It was indicatedhat no endogenous substances significantly influenced the ion-zation of the analytes.

.4.3. Linearity of calibration curves and lower limits ofuantification

The typical calibration curves for cetirizine and pseu-oephedrine were Y = 139.23X − 3.3452 (correlation coeffi-ient, r = 0.9996) and Y = 107.22 X − 1.4467 (r = 0.9998),espectively, Y: concentration in ng/ml, X: ratios of ceti-izine/pseudoephedrine peak area to that of I.S.).

The limit of quantization was 1.0 ng/ml (n = 6) for pseu-oephedrine and 1.0 ng/ml for cetirizine, and an acceptableccuracy of ±15% and a precision below 15% were obtained.his level was selected with respect to expected concentrationsf the samples from the pharmacokinetic study.

.4.4. Precision and accuracyIntra- and inter precision was assessed from the results of

Cs. The mean values and RSD for QCs at three concentrationevels were calculated over three validation days by using a one-ay analysis of variance (ANOVA). The intra- and inter-batchrecision and accuracy data for cetirizine and pseudoephedrinere summarized in Table 1. Intra-batch precision ranged between.46–6.65% and 2.5–5.99% and the inter-batch precision wereetween 3.78–9.70% and 3.90–7.66% separately.

etrizine2.046 2.031 5.99 7.66 0.73

51.60 50.53 3.60 5.48 2.0712.8 395.2 2.46 3.90 4.62

M. Ma et al. / J. Chromatogr. B 846 (2007) 105–111 109

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ig. 2. Chromatograms of (A) blank plasma sample;(B) plasma sample spiked wlasma sample 0.5 h after oral dose of one tablet. I.S. (I), pseudoephedrine (II),

ethanol precipitation. The absolute recoveries were shown toe consistent, precise and reproducible.

The results of stability experiments showed that signifi-ant degradation occurred at −20 ◦C for 5 days and after fivereeze-thaw cycles. The accuracy values at three QC levels ofseudoephedrine and cetirizine were within ±15%. The stock

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eudoephedrine (1 ng/ml), cetirizine (1 ng/ml) and I.S. (64 ng/ml); (C) volunteeretirizine (III) in human plasma samples.

olutions in water were stable at 4 ◦C for a week. The methanololution of I.S. (80 ng/ml) was proved stable at room tempera-

ure for more than 12 h and at 4 ◦C for a week. There was noignificant degradation under the conditions described in thistudy and these results indicated that analytes were stable underoutine laboratory conditions.

110 M. Ma et al. / J. Chromatogr. B 846 (2007) 105–111

Table 2Recovery data for analytes in human plasma (n = 5)

Added concentration (ng/ml) Recoverya (mean ± SD, %) RSDb (%)

Pseudoephedrine2.048 96.24 ± 8.26 8.59

102.4 91.95 ± 4.11 4.48819.2 95.81 ± 2.05 2.13Cetrizine

2.046 92.87 ± 7.11 7.6651.60 93.78 ± 4.69 5.01

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Table 3Comparison of mean pharmacokinetic parameters of single- and multiple- oraldosage administration (n = 10)

Parameter One tablet Two tablets

Mean SD Mean SD

PseudoephedrineAUC (h ng/ml) 4441.74 755.12 10404.74 1634.49Cmax (ng/ml) 350.36 57.33 701.83 57.30Tmax (h) 4.0 0.8 4.2 0.8t1/2 (h) 5.37 0.52 6.11 0.92

CetrizineAUC (h ng/ml) 1099.77 255.49 2372.24 625.73Cmax (ng/ml) 167.06 47.17 323.20 77.16Tmax (h) 0.7 0.5 0.7 0.5t1/2 (h) 6.76 1.82 6.17 1.39

Table 4Comparison of mean pharmacokinetic parameters under the fasted and fed con-ditions (n = 10)

Parameter Fasted Fed

Mean SD Mean SD

PseudoephedrineAUC (h ng/ml) 4637.30 570.01 4384.13 488.69Cmax (ng/ml) 352.99 42.98 378.74 42.18Tmax (h) 4.2 0.8 4.2 0.8t1/2 (h) 5.44 0.58 4.94 0.61

CetrizineAUC (h ng/ml) 1232.47 194.18 1153.21 207.26Cmax (ng/ml) 173.70 35.47 122.97 15.15

RSD = relative standard deviation.b Mean relative error = (overall mean assayed concentration − added concen-

ration)/added concentration ×100.

.4.6. Application of the method to a pharmacokinetictudy in healthy volunteers

The proposed method was applied to the determination oflasma concentrations and the pharmacokinetic parameters ofhe combination table were first reported in Chinese people.

The mean plasma concentration-time curve of oral admin-stration is shown in Fig. 3A The comparison of correspondingharmacokinetic parameters is listed in Table 3. The AUC, Cmaxre showing linear between one tablet and two tablets; Tmax and1/2 were not statistically different for the two drugs. And thealculated pharmacokinetic parameters of pseudoephedrine andetrizine are in agreement with literature separately [13,16].

Fig. 3B shows the profiles of the mean concentration of pseu-oephedrine and cetrizine in fed and fasted states. The collectedharmacokinetic parameters are listed in Table 4. The results ofur study demonstrated that the presence of standard meal had

o effect on absorption of pseudoephedrine, which was the sames the former report [16,17]. The mean pharmacokinetic param-ters of cetirizine had no significant statistical differences exceptor Cmax and Tmax which reduced 30% and delayed 2.4 h in the

ig. 3. Mean plasma concentration-time curve of pseudoephedrine and cetrizinet different conditions of administration. (n = 10).

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ed state. That means standard meal slightly reduce the rate butot the extent of cetirizine absorption. These results agree withreviously published reports [18,19], and the present method isn excellent analytical option for quantifying pseudoephedrinend cetrizine in human plasma.

. Conclusions

A LC–MS/MS method to determine simultaneously the con-entration of pseudoephedrine and cetrizine in human plasmaas developed. The presented was then successfully applied for

he evaluation of Phase-I pharmacokinetic study and the col-ected parameters were first reported in healthy Chinese people.he result proved that the method is sensitive, highly selectivend stable.

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